Process for producing nucleosides

ABSTRACT

The present invention concerns a process for producing nucleosides by carrying out the reaction of a base donor, a saccharide residue donor and a phosphoric acid donor by the use of an enzyme preparation containing nucleoside phosphorylase, thereby forming an N-glycosidic bond between the base moiety of the base donor and the saccharide moiety of the saccharide residue donor, which comprises using, as the enzyme preparation containing nucleoside phosphorylase, a preparation derived from the cells of one or more kinds of microorganisms belonging to thermophiles of the genus Bacillus and having high nucleoside phosphorylase activity per unit cell weight.

TECHNICAL FIELD

This invention relates to a process for producing nucleosides by the useof an enzyme preparation containing nucleoside phosphorylase derivedfrom thermophiles belonging to the genus Bacillus.

BACKGROUND ART

As processes for producing nucleosides by reacting a saccharide residuedonor such as nucleoside and ribose 1-phosphate enzymatically with abase donor, for example, there have been reported various processes forproducing various purine nucleosides (Japanese Patent Publications Nos.24475/1968, 28954/1968, 28955/1968, 28956/1968, 11116/1970, 14957/1973,Japanese Laid-Open Patent Publications Nos. 71495/1980, 18599/1981,142293/1981, 164793/1981, 166199/1981, 63393/1983, 94396/1983,170493/1983, etc.), processes for producing various pyrimidinenucleosides (Japanese Patent Publication No. 16478/1960, JapaneseLaid-Open Patent Publications Nos. 102794/1981, 213397/1984,239495/1985, etc.), and processes for producing other variousnucleosides (Japanese Laid-Open Patent Publications Nos. 29720/1975,146593/1982, 190396/1983, 216696/1983, 143599/1984, 179094/1984,213397/1984, 120981/1985, 133896/1985, 31093/1988, 177797/1988, etc.).

However, although these enzymatic processes for production ofnucleosides are excellent as compared with chemical synthetic methodswith respect to substrate specificity and steric selectivity inherent inenzymatic reaction, the activity of the enzyme has not been sufficient,and they have not been found satisfactory with respect to yield in allcases.

Also, when the reaction is carried out at room temperature, lowering inyield which may be considered to be caused by contamination withbacteria is observed, and when the reaction is carried out at a highertemperature (e.g., 45° C. or higher) in order to avoid contamination,the enzyme gradually becomes deactivated, consequently leading to amarked lowering in yield.

Generally speaking, synthesis of a compound will be brought about byinclination of the equilibrium between the formation reaction and thedecomposition reaction toward the formation reaction For this reason,for increasing the yield of the compound, it is important to promote theformation reaction and suppress the decomposition reaction, and thisprinciple is not exceptional in the enzymatic process for the productionof a compound.

Also, if the reaction temperature is made higher, the reaction rate willbecome more rapid, whereby the reaction will be completed within ashorter time and the solubility of the substrate will also be enhanced,and hence it has the possibility of producing the objective product withgood yield.

When producing nucleosides by the use of nucleoside phosphorylase, forpromoting the formation reaction of nucleosides, consideration must betaken from the two points of the activity of the enzyme itself to beused as a catalyst and the reaction conditions. Selection of thereaction conditions is no more than an auxiliary means for inducing theactivity of the enzyme employed, and the drastic method for promotingthe formation reaction to increase the yield of the objective compoundsis to use nucleoside phosphorylase having excellent activity.

The nucleoside phosphorylases of the prior art which have been used inthe production of nucleosides are for the most part those prepared frommicroorganisms which can be easily cultured. However, when the activityof the enzyme is examined from the efficiency of the reaction, specificactivity, heat resistance, yield of the objective compound, etc., thosewhich have been used in the prior art have not always been satisfactory.

On the other hand, concerning the enzyme which has been considered toparticipate in the decomposition reaction of the production ofnucleosides, for example, nucleosidase, the method of inhibiting thenucleosidase by means of the immobilization method using a photocurableresin has been reported (Japanese Laid-Open Patent Publication No.253393/1987). This method is excellent, but when using microorganismcells as an enzyme preparation, some microorganisms cannot be easilyimmobilized, thus the method lacks general purpose applicability.

The present inventors have screened various microorganisms in order todiscover enzymes of excellent activity which can be used for enzymaticproduction of nucleosides and consequently discovered a group ofmicroorganisms containing a large amount of heat-resistant nucleosidephosphorylase having extremely high specific activity, and having highnucleoside phosphorylase activity per unit cell weight amongthermophiles belonging to the genus Bacillus.

In the prior art, nucleoside phosphorylase has been isolated andpurified from Bacillus stearothermophilus which is a thermophilebelonging to the genus Bacillus and the enzymatic properties of theenzyme have been reported (see J. Biol. Chem., 244, 3691-3697 (1969),Agric. Biol. Chem., 53, 2205-2210 (Aug. 23, 1989), Agric. Biol Chem.,53, 3219-3224 (Dec. 23, 1989)). Also, a process for producing5-methyluridine or thymidine by using microorganism cells of Bacillusstearothermophilus as an enzyme source has been reported (see JapaneseLaid-Open Patent Publication No. 320995/1989 (published on Dec. 27,1989), Agric. Biol. Chem., 53, 197-202 (Jan. 23, 1989)). However, thenucleoside phosphorylases of the above-mentioned reports, althoughhaving the advantage of heat resistance, have low specific activity andalso low enzyme activity per unit cell weight and thus could not solvethe problem of the prior art that no nucleoside can be efficientlyproduced More specifically, when the yield of the nucleoside disclosedin Japanese Laid-Open Patent Publication No. 320995/1989 is representedin terms of its proportion relative to the base donor employed, it is atmost around 30% (even if the reaction has occurred ideally, the yield ofthe objective product determined from the equilibrium constant of theenzyme reaction is 53 to 56%).

DISCLOSURE OF THE INVENTION

The present inventors have further studied the group of microorganismsfound by the present inventors as mentioned above, containing a largeamount of heat-resistant nucleoside phosphorylase having high specificactivity, and having high nucleoside phosphorylase activity per unitcell weight, and consequently found that (1) the group of thesemicroorganisms, while having both purine nucleoside phosphorylase andpyrimidine nucleoside phosphorylase having heat resistance and highspecific activity in combination, contains no nucleosidase, or if any,exhibits very weak activity at the reaction temperature during theproduction of nucleosides (35° to 80° C.), and that (2) by the use ofthe enzyme preparation containing the nucleoside phosphorylase derivedfrom the cells of one or more kinds of the microorganisms in theenzymatic production of nucleosides, the objective nucleosides can beproduced with good yield within a short time only by a small amount ofthe enzyme, whereby the present invention has been accomplished.

More specifically, the present invention concerns, in a process forproducing nucleosides by carrying out the reaction of a base donor, asaccharide residue donor and a phosphoric acid donor by the use of anenzyme preparation containing nucleoside phosphorylase, thereby formingan N-glycosidic bond between the base moiety of the base donor and thesaccharide moiety of the saccharide residue donor, the improvement whichcomprises using, as the enzyme preparation containing nucleosidephosphorylase, a preparation derived from the cells of one or more kindsof microorganisms belonging to thermophiles of the genus Bacillus andhaving high nucleoside phosphorylase activity per unit cell weight.

In the present specification, "nucleosides" refer to nucleosidesexisting in nature such as uridine, thymidine, cytidine, adenosine andguanosine, and also include various nucleoside analogs.

Also, the present invention concerns the above-mentioned enzymepreparation itself, the novel microorganism to be used for preparationof said enzyme preparation, and the novel nucleoside phosphorylaseprepared from said microorganism which can be used for the production ofnucleosides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph indicating the optimum pH and the stable pH range ofthe purine nucleoside phosphorylase of the present invention.

FIG. 2 is a graph indicating the optimum temperature and the stabletemperature range of the purine nucleoside phosphorylase of the presentinvention.

FIG. 3 is a graph indicating the optimum pH and the stable pH range ofthe pyrimidine nucleoside phosphorylase of the present invention.

FIG. 4 is a graph indicating the optimum temperature and the stabletemperature range of the pyrimidine nucleoside phosphorylase of thepresent invention.

FIG. 5 is a graph indicating a comparison of the formation ratio of1-β-D-ribofuranosyl-1, 2, 4-triazole-3-carboxamide (ribavirin) relativeto the reaction time when ribavirin is produced by the use of the cellsof Bacillus stearothermophilus TH6-2 or Brevibacterium acetvlicum AT-6-7as an enzyme source.

BEST MODE FOR PRACTICING THE INVENTION Enzyme preparation containingnucleoside phosphorylase

The "enzyme preparation containing nucleoside phosphorylase" of thepresent invention (hereinafter called "enzyme preparation") refers to apreparation containing at least one, preferably both, of purinenucleoside phosphorylase and pyrimidine nucleoside phosphorylase.

The "degree of enzyme purity" means the proportion of the enzyme proteinamount in the total protein amount.

The enzyme preparation of the present invention can be prepared from themicroorganisms having high nucleoside phosphorylase activity per unitcell weight (hereinafter called "the microorganisms of the presentinvention") among the microorganisms belonging to thermophiles belongingto the genus Bacillus, specifically moderate thermophiles such asBacillus acidocaldarius, Bacillus schlegeli and Bacillusstearothermophilus.

As nucleoside phosphorylase activities for selection of themicroorganisms, for example, the following values may be mentioned as ameasure:

purine nucleoside phosphorylase

10 or more U/g-wet cells, preferably 12 or more U/g-wet cells

pyrimidine nucleoside phosphorylase

10 or more U/g-wet cells, preferably 15 or more U/g-wet cells

The microorganism which satisfies one of these two conditions can beused as a preparation source for the enzyme preparation to be used inthe process of the present invention, and the microorganism whichsatisfies the above two conditions at the same time is preferable as apreparation source for the enzyme preparation.

Specific examples of preferable microorganisms satisfying suchconditions are Bacillus stearothermophilus TH6-2, P-21, P-23, etc. (allare strains isolated from soil on the grounds of Yamasa Shoyu K.K.). Thebacteriological characteristics of the TH6-2 strain which is the mostrepresentative among such strain group are shown below.

Bacteriological Characteristics of TH6-2 (A) MorphologicalCharacteristics

(1) Shape and size of cell: Short rod, 0.6-1.1×2-7 μm

(2) Formation of spores: Positive

(3) Swelling of sporangium: Positive

(4) Site and size of spores within cell: At the terminal or the center;0.8×0.8-1.0 μm

(5) Gram staining: Gram variable (Gram-positive in the initial stage ofcultivation)

(B) Cultural Characteristics on Various Culture Media

(1) Broth agar (Bouillon-agar) slant medium: Abundant growth, smoothsurface, opaque, no medium change

(2) Broth agar (Bouillon-agar) plate medium: Circular colony formation,thinly spread, exhibiting viscous property, opaque, wavy at the brim

(3) Litmus-milk medium: No growth

(C) Physiological Properties

(1) Growth in the presence of oxygen: Grown

(2) Growth in the absence of oxygen: Not grown

(3) Catalase: Positive

(4) V-P test: Negative

(5) Methyl red test (pH in V-P broth): <pH 6

(6) Hydrolysis:

Casein: Negative

Gelatin: Positive

Starch: Positive

(7) Utilization of citric acid: Positive

(8) Reduction of nitrate: Positive

(9) Formation of indole: Negative

(10) Requirement of sodium chloride or potassium

chloride: Negative

(11) Formation of acids from saccharides:

Positive glucose, arabinose, xylose, fructose, maltose

Negative: starch, glycerol, sucrose, raffinose

(12) Growth at respective pH's:

Grown at pH 6.8, not at pH 5.7

(13) Growth in the presence of sodium chloride

in the presence of 2% NaCl: Grown

in the presence of 5% NaCl: Not grown

(14) Growth range:

Growth pH range: 6.5 to 9.0

Growth temperature range: 35° to 65° C.

(15) Growth in the presence of glucose:

Not grown in the presence of 0.5% or more of

glucose

By referring these bacteriological characteristics to the classificationstandards of Bergey's Manual of Systematic Bacteriology (EighthEdition), the above microorganism was found to belong to Bacillusstearothermophilus and designated as Bacillus stearothermophilus TH6-2.P-21 and P-23 were also found to exhibit the same bacteriologicalcharacteristics. TH6-2 has been deposited with the Fermentation ResearchInstitute, Agency of Industrial Science and Technology 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan in conformity with theBudapest treaty, and has been given FERM BP-2758 as the depositionnumber. This international deposition was based on the transference madeon Feb. 16, 1990 from FERM P-10526 deposited domestically at the abovedepository on Feb. 4, 1989.

TH6-2, P-21 and P-23 all belong to Bacillus stearothermophilus, and canbe distinguished clearly from known microorganisms in having very highnucleoside phosphorylase activity per unit cell weight and containingsubstantially no nucleosidase. For example, the comparison test ofpyrimidine nucleoside phosphorylase and purine nucleoside phosphorylaseactivities per unit cell weight among known Bacillus stearothermophilusmicroorganisms stored at American Type Culture Collection (ATCC) and theabove microorganisms of the present invention gave the results as shownin Table 1. From Table 1, it can be seen that the nucleosidephosphorylase activities per unit cell weight of TH6-2, P-21 and P-23are all higher by a little less than 2-fold for purine nucleosidephosphorylase activity and by a little more than 6-fold for pyrimidinenucleoside phosphorylase activity as compared with the enzyme activitiesexhibited by the strains known in the art. Specifically, the presentmicroorganisms were found to exhibit purine nucleoside phosphorylaseactivity and pyrimidine nucleoside phosphorylase activity per unit cellweight of 13-15 U/g-wet cells and 20-22 U/g-wet cells, respectively.

                  TABLE 1                                                         ______________________________________                                                       Purine      Pyrimidine                                                        Nucleoside  Nucleoside                                                        Phosphorylase                                                                             Phosphorylase                                                     Activity    Activity                                           Microorganism  (U/g-wet cells)                                                                           (U/g-wet cells)                                    ______________________________________                                        Microorganisms of the                                                         present invention                                                             (TH-6-2)       14.2        2.8                                                (P-21)         13.9        21.8                                               (P-23)         13.4        21.4                                               ATCC 8005      2.3         1.4                                                ATCC 10149     5.4         0.8                                                ATCC 12016     2.6         1.4                                                ATCC 12976     2.3         0.4                                                ATCC 12978     2.8         0.8                                                ATCC 12980     4.5         0.4                                                ATCC 15952     1.2         0.8                                                ATCC 21365     8.7         3.4                                                ATCC 29609     3.4         0.4                                                ______________________________________                                    

Thus, the microorganisms of the present invention were found tosufficiently satisfy the selection standards of nucleoside phosphorylaseactivity as described above and to be useful as a preparation source ofthe enzyme preparation to be used for the production of nucleosides.

For verifying this fact, ribavirin was produced and the formation ratioof the objective compound was compared among the present microorganismsand known strains. As a result, as shown in Table 2, while formationratios were 90% or more when all of the microorganisms of the presentinvention were employed, the formation ratio of known microorganisms wasonly 40% at the highest, whereby it was confirmed that themicroorganisms of the present invention are extremely useful as anenzyme source for the production of nucleosides.

                  TABLE 2                                                         ______________________________________                                                       Formation Ratio of                                                            Ribavirin (%) <Ratio                                                          Relative to 1,2,4-Triazole-                                    Microorganism  3-Carboxamide>                                                 ______________________________________                                        Microorganisms of the                                                         present invention                                                             (TH-6-2)       95.0                                                           (P-21)         95.0                                                           (P-23)         94.1                                                           ATCC 7953      3.8                                                            ATCC 8005      30.3                                                           ATCC 10149     11.7                                                           ATCC 12016     44.4                                                           ATCC 12976     8.0                                                            ATCC 12978     43.5                                                           ATCC 12980     0                                                              ATCC 15952     39.0                                                           ATCC 21365     37.6                                                           ATCC 29609     4.1                                                            ______________________________________                                    

In the comparison test as described above, the cells of microorganismswere prepared in the same manner as in Example 1 described below, andpurine nucleoside phosphorylase activity and pyrimidine nucleosidephosphorylase activity were assayed according to the methods as alsodescribed below. On the other hand, ribavirin was produced by adding 10ml of a suspension of microorganisms with equal cell weight (containing200 mg as wet cells ) to 10 ml of a substrate solution (aqueous solutionof pH 6.0 containing 40 mM 1, 2, 4-triazole-3-carboxamide, 60 mMuridine, 40 mM potassium dihydrogenphosphate) and stirring the mixtureof 50° C. for 24 hours. After the above reaction, the reaction mixturewas centrifuged, the supernatant was diluted to 50 to 100-fold, and thiswas subjected to the HPLC method (column: YMC A-312 (manufactured byYamamura Kagaku Kenkyusho, K.K.), eluent: 0.15M potassiumdihydrogenphosphate solution, detection: absorbance at 220 nm) for themeasurement of the amount of ribavirin formed.

The formation ratio was determined from the following formula: ##EQU1##

The enzyme preparation of the present invention can be prepared byculturing a microorganism belonging to the microorganism group of thepresent invention and suitably processing the microorganism cellsobtained by the cultivation according to a use mode corresponding to usepurpose.

As a medium for culturing the microorganism, those containingappropriate amounts of carbon sources and nitrogen sources assimilableby the microorganism, and containing also, if necessary, metal salts,trace amount growth promoting substances, defoaming agents, etc. addedtherein are employed. More specifically, examples of the mediumcomponents are saccharides (glucose, saccharose, etc.), naturalcarbohydrates (molasses, waste molasses, starch, wheat, bran, rice,etc.), alcohols, fatty acids, hydrocarbons, etc., and as nitrogensources, meat extract, yeast extract, soybean hydrolyzate, Casaminoacid, various amino acids, urea, etc., as inorganic salts, phosphates,hydrochlorides and sulfates of metals such as zinc, iron, magnesium,sodium, calcium, potassium, etc., and as trace amount growth promotingsubstances, vitamin B₁, vitamin B₂, pantothenic acid, and biotin.

Cultivation is carried out according to a conventional liquid culturemethod (shaking culture, aeration stirring culture, stationary culture,continuous culture, etc.)

The culture conditions depend on the microorganism and the culturemedium employed, and thus cannot be specified. Generally, the pH at theinitiation of culture is adjusted to 6.5 to 9.0, and cultivation iscarried out until the desired enzyme activity can be amply obtainedunder the temperature condition of about 35° to 65° C., specifically forabout 5 to 50 hours.

The mode of the enzyme preparation prepared by the use of the culturebroth containing viable microorganisms thus obtained (hereinafter calledcultured product) is not particularly limited, but, for example, thecultured product itself of the microorganisms, the viable microorganismcells separated from the cultured product by a conventional separationmethod (centrifugation, precipitation, agglutination, washing, etc.), orthe treated product of the cells may be mentioned.

Further specific examples of the treated product of viable cells are thedestroyed products of viable cells and the viable organisms with thecell walls and/or the cell membranes having been denatured obtained bytreatment of the viable cells by the treatment methods used in generalsuch as mechanical destruction (by Waring blender, French press,homogenizer, crucible, etc.), freezing and thawing, drying (freezedrying, air drying, acetone drying, etc.), autolysis (by solventtreatment with toluene, ethyl acetate, etc.), enzyme treatment (by cellwall dissolving enzyme such as lysozyme), sonication, osmotic pressureshock and chemical treatment (with solution of salts, acidic solution,alkaline solution, surfactant, chelating agent, etc.); or crude enzymeor purified enzyme obtained by fractionating the fractions having enzymeactivity and further treating the extracted fractions, if necessary,according to the general enzyme purification methods (salting out,isoelectric point precipitation, organic solvent precipitation, variouschromatographies, dialysis) to fractionate the fractions having thedesired enzyme activity of the present invention.

Such a cultured product, viable microorganism cells and treated productmay be used in the free state without applying immobilization treatmentthereto, or may be used as an immobilized product obtained by animmobilization treatment such as entrapping, crosslinking or adsorption.

A specific example of the purified enzyme which is one form of thetreated product of cells is the nucleoside phosphorylase obtained byextraction/purification from Bacillus stearothermophilus TH6-2 belongingto the microorganism group of the present invention having enzymologicalproperties as described below.

(A) Purine Nucleoside Phosphorylase

(1) Action

    Purine Nucleoside+Phosphoric Acid ⃡Purine Base+Pentose 1-Phosphate

The purine nucleoside phosphorylase of the present invention catalyzesthe above phosphorolysis. For this reason, it belongs to theInternational Enzyme Classification E.C.2.4.2.1.

(2) Substrate specificity

The results of phosphorolysis of various purine nucleoside substratesare shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                          Amount of Base                                                                            Relative                                                          Formed      Activity                                        Substrate         (μmol 10 min.)                                                                         (%)                                             ______________________________________                                        Adenosine         0.15        5                                               2'-Deoxyadenosine 0           0                                               3'-Deoxyadenosine 0           0                                               Arabinofuranosyl adenine                                                                        0           0                                               Inosine           2.55        100                                             2'-Deoxyinosine   2.54        100                                             3'-Deoxyinosine   0           0                                               Guanosine         2.15        84                                              2'-Deoxyguanosine 2.18        85                                              ______________________________________                                    

From Table 3, it can be seen that the purine nucleoside phosphorylase ofthe present invention is specific for inosine, 2'-deoxyinosine,guanosine and 2'-deoxyguanosine within the range tested.

(3) Optimum pH and pH stability

The optimum pH is pH 7 to 8, and the stable pH range is pH 5 to 9 (seeFIG. 1).

(4) Optimum temperature and temperature stability

The optimum temperature is 60° to 80° C., and the stable temperaturerange is up to 60° C. (see FIG. 2).

(5) Molecular weight

The molecular weight measured by SDS-polyacrylamide gel electrophoresisis about 31,000.

(6) Titre (specific activity)

At 80% of enzyme purity, the specific activity of 400 or more (U/mg) isexhibited, and at 90% of enzyme purity, 450 (U/mg).

(B) Pyrimidine Nucleoside Phosphorylase

(1) Action

    Pyrimidine Nucleoside+Phosphoric Acid ⃡Pyrimidine Base+Pentose 1-Phosphate

The pyrimidine nucleoside phosphorylase of the present inventioncatalyzes the above phosphorolysis. For this reason, it belongs to theInternational Enzyme Classification E.C.2.4.2.2.

(2) Substrate specificity

The results of phosphorolysis of various pyrimidine nucleosidesubstrates are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                         Amount of Base                                                                            Relative                                                          Formed      Activity                                         Substrate        (μmol/10 min.)                                                                         (%)                                              ______________________________________                                        Uridine          1.73        68                                               2'-Deoxyuridine  2.55        100                                              Arabinofuranosyl uracil                                                                        0           0                                                Pseudouridine    0           0                                                Cyclouridine     0           0                                                Cytidine         0           0                                                2'-Deoxycytidine 0           0                                                Ribofuranosyl thymine                                                                          0.96        38                                               Thymidine        1.71        67                                               ______________________________________                                    

From Table 4, it can be seen that the pyrimidine nucleosidephosphorylase of the present invention is specific for uridine,2'-deoxyuridine, ribofuranosyl thymine, thymidine within the rangetested.

(3) Optimum pH and pH stability

The optimum pH is pH 7 to 9, and the stable pH range is pH 5 to 9 (seeFIG. 3).

(4) Optimum temperature and temperature stability

The optimum temperature is 60° to 70° C., and the stable temperaturerange is up to 60° C. (see FIG. 4).

(5) Molecular weight

The molecular weight measured by SDS-polyacrylamide gel electrophoresisis about 45,000.

(6) Titre (specific activity)

At 80% of enzyme purity, the specific activity of 250 or more (U/mg) isexhibited, and at 90% of enzyme purity, 297 (U/mg).

The above enzymatic properties were assayed according to the methodsdescribed below.

1. Assay of Activity Purine Nucleoside Phosphorylase Activity

Into 1.0 ml of a substrate solution (aqueous solution of pH 8.0containing 20 mM inosine and 0.1M potassium dihydrogenphosphate) isadded 20 μl of an enzyme solution (50 mM acetate buffer (pH 6.0)containing 1 μg of purified enzyme), and the reaction is carried out at50° C. for 10 minutes. Then the reaction is stopped by addition of HC1to a final concentration of 0.1N, and also cooling at 0° C. for 10minutes. Next, the reaction is subjected to centrifugation, and thesupernatant obtained is then subjected to the HPLC method (column: YMCA-312 (manufactured by Yamamura Kagaku Kenkyusho, K.K.), eluent: 20 mMTris-HCl buffer (pH 7.5) containing 5.0% acetonitrile, detection: 260nm) to quantitate the hypoxanthine formed. The enzyme amount forming 1μmol of hypoxanthine per one minute is defined as one unit ("U").

Pyrimidine Nucleoside Phosphorylase Activity

Except for using uridine in place of inosine in the substrate solutionand quantitating uracil according to the HPLC method, the same method asin the above-described assay of purine nucleoside phosphorylase activityis conducted. The amount of enzyme forming 1 μmol of uracil per oneminute is defined as one unit.

2. Substrate Specificity

By the use of an aqueous solution of pH 8.0 containing 10 mM of anucleoside and 50 mM of potassium dihydrogenphosphate as a substratesolution, the reaction is carried out at 50° C. for 10 minutes, andafter the reaction, except for quantitating the base of the nucleosideaccording to the HPLC method, the same method as in the assay of purinenucleoside phosphorylase activity is conducted.

3. Optimum pH

The same method as in the assay of purine nucleoside phosphorylaseactivity is conducted except for using a substrate solution prepared bydissolving a nucleoside (20 mM inosine or uridine) and 0.1M potassiumdihydrogenphosphate, and adjusting to pH 4 to 10 with a diluted aqueoussolution of hydrochloric acid or sodium hydroxide.

4. Stable pH

Except for using an enzyme solution incubated in a 0.2M acetate buffer(pH 3.5 to 6) and a Tris-HCl buffer pH 7 to 9) at 37° C. for 16 hours,the same method as in the assay of either purine nucleosidephosphorylase activity or pyrimidine nucleoside phosphorylase activityis conducted.

5. Optimum Temperature

Except for carrying out the reaction at the respective temperatures of30° to 80° C., the same method as in the assay of either purinenucleoside phosphorylase activity or pyrimidine nucleoside phosphorylaseactivity is conducted.

6. Stable Temperature Range

Except for using an enzyme solution heated at 30° to 80° C. for 15minutes, the same method as in the assay of either purine nucleosidephosphorylase activity or pyrimidine nucleoside phsophorylase activityis conducted.

The characteristics of the enzyme obtained from the microorganism of thepresent invention as described above are that it has optimum temperatureand stable temperature ranges at relatively higher temperatures and alsohas a markedly high specific activity. Therefore, by the use of apreparation containing an enzyme having such characteristics for theproduction of nucleosides, the amount of the enzyme preparation to beused for the reaction can be made small, whereby the nucleosides can beprepared with good yield by the use of a small amount of enzyme.Further, since the reaction can be carried out at a relatively highertemperature (45° C. or higher), contamination with bacteria can be alsoprevented.

II. PRODUCTION OF NUCLEOSIDES

The production of nucleosides by the use of the enzyme preparation asdescribed above can be practiced by carrying out in a reactor thecontact reaction of a base donor, a saccharide residue donor and aphosphoric acid donor as described below with the enzyme preparation.

(1) Base Donor

The base donor to be used in the process of the present invention is acompound which can supply a base into the reaction system. The basedonor to be used is selected according to the objective nucleoside,examples being heterocyclic bases and derivatives thereof capable offorming an N-glycoside bond with the saccharide moiety of the saccharideresidue donor through the action of nucleoside phosphorylase. Specificexamples of heterocyclic bases are purine and derivatives thereof,pyrimidine and derivatives thereof, triazole and derivatives thereof,imidazole and derivatives thereof, deazapurine and derivatives thereof,azapyrimidine and derivatives, or pyridine and derivatives thereof. Asthe base donor, heterocyclic bases themselves as a matter of course, andalso nucleosides and nucleotides having said heterocyclic bases may beemployed.

Specifically, examples are purine derivatives having substituents at 1or more positions of the 1-position, 2-position, 6-position or8-position of the purine base (e.g., amino group, substituted aminogroup, hydroxyl group, oxo group, mercapto group, acyl group, alkylgroup, substituted alkyl group, alkoxyl group and halogen atom), such asadenine, guanine, hypoxanthine, xanthine, 6-mercaptopurine,6-thioguanine, N⁶ -alkyl or acyladenine, 2-alkoxyadenine, 2-thioadenineand 2, 6-diaminopurine; pyrimidine derivatives having the samesubstituents as mentioned above at 1 of more positions of the 2position, 4-position or 5-position of pyrimidine, such as cytosine,uracil, thymine, 5-halogenouracil (5-fluorouracil and 5-iodouracil),5-halogenocytosine (5-fluorocytosine), 5-trihalogenomethyluracil(5-trifluoromethyluracil), 2-thiocytosine, 4-thiouracil, N⁴-acylcytosine and 5-halogenovinyluracil; 1, 2, 4-triazole derivativeshaving a substituent at the 3-position of 1, 2, 4-triazole, such as 1,2, 4-triazole-3-carboxamide, 1, 2, 4-triazole-3-carboxylic acid and 1,2, 4-triazole-3-carboxylic acid alkyl ester; imidazole derivativeshaving substituents at the 4-position or 5-position of imidazole, suchas 5-aminoimidazole-4-carboxamide, 4-carbamoyl-imidazolium-5-oleate andbenzimidazole; deazapurine derivatives at the 1-position, 3-position or7-position of purine, such as 1-deazadenine, 3-deazadenine,3-deazaguanine, 7-deazadenine, 7-deazaguanine, or compounds having thesame substitutes as in the above-mentioned purine derivatives; azapurinederivatives such as 8 -azadenine and 7-deaza-8-azahypoxanthine(allopurinol); azapyrimidine derivatives such as 5-azathymine,5-azacytosine and 6-azauracil; pyridine derivatives such as3-deazauracil, nicotinic acid and nicotinic acid amide.

Saccharide Residue Donor

The saccharide residue donor is for supplying a saccharide residue intothe reaction system. That is, the saccharide residue donor is selectedaccording to the objective nucleoside, and ribose compounds anddeoxyribose compounds capable of forming an N-glycosidic bond with thebase moiety of the base donor through the action of nucleosidephosphorylase are examples. Examples of ribose compounds areribonucleosides such as inosine, guanosine, uridine andribofuranosylthymine, and ribose 1-phosphate, while examples ofdeoxyribose compound are deoxynucleosides such as 2'-deoxyinosine,2'-deoxyguanosine, 2'-deoxyuridine, thymidine, 2', 3'-dideoxyinosine,2', 3'-dideoxyguanosine, 2', 3'-dideoxyuridine and 3'-deoxythymidine,and 2-deoxyribose 1-phosphate and 2, 3-dideoxyribose 1-phosphate.

When as an enzyme preparation, a preparation other than a purifiedenzyme is to be used, in addition to the saccharide residue donormentioned above, ribose compounds such as adenosine, cytidine andxanthosine, and deoxyribose compounds such as 2'-deoxyadenosine,2'-deoxycytidine and 2'-deoxyxanthosine can be further used.

(3) Phosphoric Acid Donor

As the phosphoric acid donor, any compound dissociable into phosphateions in the reaction mixture may be used. For example, free phosphoricacid or phosphates (e.g., alkali metal salts such as sodium andpotassium, and ammonium salts) are preferably used. Also, as thephosphoric acid donor, a system capable of liberating phosphate ions inthe reaction mixture, for example, combinations of various phosphoricacid ester derivatives and phosphatase, combinations of nucleotides andnucleotidase can also be utilized.

(4) Reaction Conditions

As the reaction mixture, a mixture containing a base donor, a saccharideresidue donor and a phosphoric acid donor dissolved or suspended inwater or a buffer is employed. By bringing the reaction mixture intocontact with the enzyme preparation as described above, the nucleosidecorresponding to the base donor employed is produced enzymatically.

The concentrations of the base donor, the saccharide residue donor andthe phosphoric acid donor employed are suitably selected from the rangeof 0.1 to 500 mM.

The reaction generally proceeds well at a temperature ranging from 35°to 80° C., but particularly a reaction temperature of about 40° to 70°C. is preferred. If the reaction temperature is 35° C. or less, thereaction rate is slow and gives rise to poor reaction efficiency. On theother hand, at a reaction temperature of 80° C. or higher, there is therisk of the nucleoside phosphorylase activity being lowered.

The pH of the reaction mixture is generally maintained in the range ofpH 5 to 10, preferably pH 5 to 9. When the pH changes during thereaction, it may be corrected to a preferable pH range by the use of anacid or an alkali.

After the reaction, the reaction mixture is separated from the enzymepreparation and subjected to the separation/purification step of theobjective nucleoside.

The nucleoside thus formed can be separated and purified according to aknown method or its modified method. For example, variouschromatographies such as ion exchange chromatography, adsorptionchromatography, partition chromatography and gel filtration; the methodsutilizing partition between two liquid phases such as countercurrentdistribution and countercurrent extraction; and the methods utilizingdifference in solubility such as concentration, cooling and organicsolvent addition, either alone or in a suitable combination thereof canbe used.

EXAMPLES

The present invention will now be described in specific detail withrespect to the following Examples and Comparative Examples.

EXAMPLE 1

Into 500 ml of a sterilized medium (pH 7.0) containing 0.5% of yeastextract (Difco), 1.0% of peptone (Difco), 0.7% of meat extract (Difco)and 0.3% of sodium chloride was inoculated Bacillus stearothermophilusTH6-2 (FERM BP-2758), and shaking culture was carried out at 50° C. for18 hours.

The culture broth obtained was centrifuged, and the microorganism cellswere collected and washed, and the sterilized water was added to prepare250 ml of a cell suspension. To each 10 ml of aliquot of the cellsuspension was added 10 ml of a substrate solution (pH 6.0) containing a40 mM base donor, a 40 mM saccharide residue donor and 40 mM potassiumdihydrogenphosphate, and the reaction was carried out under stirring at40° to 60° C.

After the reaction, the amounts of various nucleosides were assayed byhigh performance liquid chromatography (column: YMC A-312 (manufacturedby Yamamura Kagaku Kenkyusho, K.K.), eluent: 20 mM Tris-HCl buffer (pH7.5) containing 2.5 to 5% acetonitrile, detection: absorbance at 250-260nm). ##EQU2##

The results are shown in Table 5.

                                      TABLE 5                                     __________________________________________________________________________                              Reaction Conditions                                                                     Formation Ratio                           Base Saccharide Residue   Temperature                                                                          Time                                                                             (%)                                       Donor                                                                              Donor     Product    (°C.)                                                                         (hr)                                                                             (Relative to Base)                        __________________________________________________________________________    Adenine                                                                            Uridine   Adenosine  40     24 61                                        "    "         "          50      2 45                                        "    "         "          50     24 82                                        "    "         "          60      2 60                                        "    "         "          60     24 85                                        Adenine                                                                            2-Deoxyuridine                                                                          2'-Deoxyadenosine                                                                        40      2 25                                        "    "         "          50      2 55                                        "    "         "          60      2 63                                        "    "         "          60      4 70                                        "    "         "          60      8 78                                        "    2',3'-Dideoxyuridine                                                                    2',3'-Dideoxyadenosine                                                                   55     24  15.sup.1)                                Thymine                                                                            Uridine   Ribofuranosylthymine                                                                     40      2 48                                        "    "         "          50      2 58                                        "    "         "          60      2 60                                        "    "         "          60     24 65                                        Thymine                                                                            2-Deoxyuridine                                                                          Thymidine  40      2 23                                        "    "         "          50      2 55                                        "    "         "          60      2 66                                        "    "         "          60      8 67                                        __________________________________________________________________________     .sup.1) shown in ratio to saccharide residue donor                       

EXAMPLE 2

By using a substrate solution (pH 6.0) containing 20 mM allopurinol asthe base donor, 30 mM uridine as the saccharide residue donor and 75 mMpotassium dihydrogenphosphate, the reaction was carried out in the samemanner as in Example 1 at 60° C. for 8 hours to produce a ribofuranosylderivative of allopurinol in a yield of 95% (ratio relative toallopurinol).

EXAMPLE 3

By using a 20 mM base donor (allopurinol, benzimidazole,6-mercaptopurine, purine, 6-thioguanine), a 30 mM saccharide residuedonor (uridine, 2'-deoxyuridine) and 30 mM potassiumdihydrogenphosphate, the reaction was carried out in the same manner asin Example 1 at 50° C. for 8 hours to produce ribofuranosyl derivativesor 2'-deoxyribofuranosyl derivatives bearing various base donors as thebase. The results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                                                Formation Ratio (%)                                               Saccharide  (Ratio Relative to                                    Base Donor  Residue Donor                                                                             Base)                                                 ______________________________________                                        Allopurinol Uridine     80                                                    "           2'-Deoxyuridine                                                                           97                                                    Benzimidazole                                                                             Uridine     75                                                    "           2'-Deoxyuridine                                                                           86                                                    6-Mercaptopurine                                                                          Uridine     50                                                    "           2'-Deoxyuridine                                                                           43                                                    Purine      Uridine     94                                                    "           2'-Deoxyuridine                                                                           65                                                    6-Thioguanine                                                                             Uridine      9                                                    "           2'-Deoxyuridine                                                                           65                                                    ______________________________________                                    

EXAMPLE 4

By using a substrate solution (pH 6.0) containing 40 mM 1, 2,4-triazole-3-carboxamide (hereinafter called "triazole") as the basedonor, 40 mM uridine, inosine, cytidine, adenosine or guanosine as thesaccharide residue donor and 40 mM potassium dihydrogenphosphate,ribavirin was produced in the same manner as in Example 1. The resultsare shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                Reaction Conditions                                                                        Formation Ratio (%)                                      Saccharide                                                                              Temperature Time   (Ratio Relative to                               Residue Donor                                                                           (°C.)                                                                              (hr)   Triazole)                                        ______________________________________                                        Uridine   60           4     94.2                                             Inosine   67          48     83.6                                             Cytidine  50          24     92.9                                             Adenosine 67          24     72.7                                             Guanosine 67          24     98.4                                             ______________________________________                                    

Concerning cytidine and adenosine, the nucleoside phosphorylase of thepresent invention does not recognize them as the substrate, andtherefore it may be considered that they may be converted by co-existingdeaminase to uridine and inosine, respectively, which are then utilizedas the substrate.

EXAMPLE 5

By using a substrate solution (pH 6.0) containing 40 mM triazole as thebase donor, 60 mM inosine as the saccharide residue donor and 40 mMpotassium dihydrogenphosphate, reactions were carried out in the samemanner as in Example 1 at respective reaction temperature (40° to 70°C.) for 24 hours to produce ribavirin. The results are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                        Reaction                                                                      Temperature                                                                   (°C.)                                                                              40     50     60   63   65   67   70                              ______________________________________                                        Formation Ratio                                                                           37.0   58.0   71.7 73.7 76.0 78.7 77.9                            (%) (Ratio Relative                                                           to Triazole)                                                                  ______________________________________                                    

EXAMPLE 6

A culture product obtained in the same manner as in Example 1 wascentrifuged to obtain viable cells. Next, 2.0 g of the viable cells weresuspended in 1.0 ml of a 0.1M Tris-HCl buffer (pH 7.0), and the abovecell suspension was added into a separately prepared resin solutioncontaining 0.08 g benzoin ethyl ether as a photopolymerization initiatoradded into 8.0 g photocurable resin (ENT-2000: Kansai Paint K.K.). Afterthorough mixing, the mixture was cast onto a transparent film, which wasirradiated with rays at around 360 nm simultaneously on both the frontand back surfaces of the film for 3 minutes to obtain a photopolymer.

From the immobilized product, a portion containing 0.2 g of cells wassampled, broken into pieces, and the above immobilized product wasintroduced into 10 ml of a substrate solution (pH 6.0) containing 40 mMtriazole, 40 mM uridine and 60 mM potassium dihydrogenphosphate.Ribavirin was produced while stirring the mixture at 60° C. for 8 hours.

As a result of assay of the formation ratio according to the HPLC methodas described above, the formation ratio of ribavirin relative totriazole was found to be 90%.

Further, when the reaction was carried out repeatedly 10 times, theformation ratio of ribavirin was maintained at 90% and no lowering inenzyme activity was observed.

EXAMPLE 7

Into the cultured product obtained similarly as described in Example 1were added triazole, uridine or inosine, and potassiumdihydrogenphosphate each to the final concentration of 40 mM, andfurther shaking culture was carried out at 50° C. (when using uridine)or 65° C. (when using inosine) for 24 hours. After cultivation, thecentrifuged supernatant was subjected to the assay of the formationratio of ribavirin by the HPLC method, and consequently the formationratio of ribavirin when using uridine was found to be 91.9% in terms ofthe ratio relative to triazole, and 65.9% when using inosine.

EXAMPLE 8 Preparation of Purified Enzyme

Bacillus stearothermophilus TH6-2 was inoculated from a bouillon slantinto a large test tube containing 10 ml of a medium adjusted to pH 7.2containing 1.0% peptone, 0.7% meat bouillon, 0.5% yeast extract and 0.3%sodium chloride and cultured overnight at 50° C. The cultured productobtained was transferred into a flask of 300 ml volume containing 30 mlof a medium of the same composition and the same pH, cultured at 50° C.for 8 hours, and by the use of the cultured product as the seed culture,the whole amount was added into a jar fermenter of 5-liter volumecontaining 3 liters of the above-mentioned medium. Then cultivation wascarried out under the conditions of 50° C., stirring speed of 350r.p.m., and aeration of 1.0 v.v.m. for 18 hours. From the culture broththus obtained, about 30 g of wet cells were obtained, which weresuspended in 1.5 liters of a 50 mM Tris-HCl buffer (pH 7.2) containing0.1% Triton X-100 (Sigma) and 5 mM EDTA. 750 mg of lysozyme (Sigma) wasadded, and the mixture was incubated at 37° C. for one hour. The lysissolution was centrifuged at 8,000 r.p.m., and the cell residue wasremoved. Thereafter the mixture was adjusted to pH 6.0 with the additionof 2N HCl and subjected to a heat treatment at 50° C. for 5 minutes,followed by centrifugation at 8,000 r.p.m. to obtain a supernatant asthe crude enzyme solution.

The crude enzyme solution was fractionated according to salting out byusing ammonium sulfate, and the protein precipitates at 40% to 90%saturation were dissolved in a 50 mM acetic acid-sodium acetate buffer(pH 6.0) and dialyzed overnight against a large amount of the samebuffer. The internal solution obtained was centrifuged to remove theprecipitates formed during dialysis. The supernatant was passed througha DEAE Toyopearl column (Toso K.K.) (2.2×60 cm) equilibrated with theabove-mentioned acetate buffer (hereinafter referred to as buffer A),and the proteins adsorbed were eluted by the linear gradient method of 0to 0.5M sodium chloride (by using buffer A), and the purine nucleosidephosphorylase fraction and the pyrimidine nucleoside phosphorylasefraction were respectively recovered. The respective active fractionswere dialyzed against buffer A and column chromatographed according tothe same procedures as described above by the use of 20 ml of DEAEToyopearl resin in a 25 ml volume syringe (Terumo K.K.), and therespective active fractions were recovered. Next, these active fractionswere respectively subjected to gel filtration by the use of ToyopearlHW-55S (Toso K.K.) column (2.4×80 cm) equilibrated with buffer A torecover both enzymes as substantially uniform purified preparations.

The protein amounts, the total activities and the degree of enzymepurity of the active fractions during the purification process of theboth enzymes are shown in Table 9.

The degree of enzyme purity was determined according to the method inwhich the relative proportions of the respective bands were measured bymeasuring the electrophoresis pattern obtained by the SDS-polyacrylamidegel electrophoresis by means of a densitometer.

                                      TABLE 9                                     __________________________________________________________________________                                                 Pyrimidine                                             Purine Nucleoside      Nucleoside                                             Phosphorylase          Phosphorylase                                          Degree of                                                                Protein                                                                            Enzyme Total                                                                              Specific                                                                            Protein                                                                            Purification                                                                         Total                                                                              Specific                              Amount                                                                             Purity Activity                                                                           Activity                                                                            Amount                                                                             Degree Activity                                                                           Activity             Fraction Name    (mg) (%)    (U)  (U/mg)                                                                              (mg) (%)    (U)  (U/mg)               __________________________________________________________________________    Crude enzyme solution                                                                           ○1                                                                         --     97700                                                                              16.1   ○1                                                                         --     87800                                                                              14.4                 (after heat treatment)                                                        Ammonium sulfate fraction                                                                       ○2                                                                         --     79500                                                                              32.0   ○2                                                                         --     81600                                                                              26.6                 (40-90% saturation)                                                           DEAE Toyopearl chromatography                                                                  400  --     66800                                                                              167   291  --     57500                                                                              198                  (first)                                                                       DEAE Toyopearl chromatography                                                                  109  74     40000                                                                              370   160  69     36100                                                                              225                  (second)                                                                      Toyopearl HW-55S  74  93     34300                                                                              465    81  90     24200                                                                              295                  chromatography                                                                __________________________________________________________________________      ○1  Total protein amount is 6080 mg                                    ○2  Total protein amount is 3770 mg                              

COMPARATIVE EXAMPLE 1

From each slant of Brevibacterium acetylicum AT-6-7 (ATCC 39311) andBacillus stearothermophilus TH6-2, the microorganism was inoculated intoa large test tube containing 10 ml of a medium (containing 1% peptone,0.7% meat extract, 0.3% sodium chloride, 0.5% yeast extract, pH 7.2),and respectively subjected to shaking culture at 28° C. (in the case ofAT-6-7) and at 50° C. (in the case of TH6-2) for 18 hours, followed bycentrifugal separation of the respective culture broths to separate themicroorganism cells. After washing cells of the same wet weight wereeach suspended in 10 ml of deionized water.

Into each of the cell suspensions was added 10 ml of a substratesolution containing 0.4 mM triazole, 0.4 mM uridine and 0.4 mM potassiumdihydrogenphosphate (adjusted to pH 7.0 in the case of AT-6-7, pH 6.0 inthe case of TH6-2), and the reaction was carried out under stirring in aclosed system at 45° C. or 65° C. Sampling was performed periodically tomeasure the formation ratio of ribavirin according to the HPLC method asdescribed above.

The results are shown in FIG. 5. As is apparent from FIG. 5, when theenzyme preparation prepared from the microorganism of the presentinvention is used, it has been found that the objective compound can beproduced in an even shorter time than AT-6-7 which was an extremelyexcellent enzyme preparation source, and the amount of the enzymepreparation used can be made smaller.

As described above, the enzyme preparation containing the nucleosidephosphorylase of the present invention contains a large amount ofnucleoside phosphorylase having high specific activity and heatresistance, is derived from the cells of one or more kinds ofmicroorganisms of the microorganism group belonging to thermophiles ofthe genus Bacillus having high nucleoside phosphorylase activity perunit cell weight, and by the use of such enzyme preparation for theproduction of nucleosides, a very useful method which has the followingspecific features and is rich in practical applicability can beprovided.

(1) A large amount of nucleoside phosphorylase with high specificactivity is contained in the enzyme preparation, and when it is employedin the production of nucleosides, nucleosides can be produced in a goodyield with a small amount of the enzyme.

(2) Nucleoside phosphorylase with optimum temperature and stabletemperature ranges in a relatively higher temperature region iscontained in the enzyme preparation. For this reason, the reaction canbe carried out at a high temperature, whereby deactivation of theenzyme, decomposition of the reaction product, etc. due to contaminationwith bacteria can be suppressed.

(3) A preparation containing both enzymes of purine nucleosidephosphorylase and pyrimidine nucleoside phosphorylase as the nucleosidephosphorylase can also be obtained. By the use of such preparation forthe nucleoside production, for example, the two enzymes will actconcomitantly as shown in the reaction scheme shown below, wherebynucleosides can be produced at a rate of 2-fold or more as compared withthe enzyme preparation containing only one nucleoside phosphorylase.##STR1##

Also, the microorganism of the present invention can grow at arelatively higher temperature, resulting in a rapid growth rate, andcontains a large amount of the enzymes suitable for the nucleosideproduction as described above in the cells obtained by cultivation.Therefore it is extremely useful as an enzyme preparation or apreparation source therefor to be used for the nucleoside production.

Further, by the use of the cultured product of the microorganism as anenzyme preparation of the present invention, autolysis of the cells canbe prevented.

Also, the nucleoside phosphorylase obtained by the microorganism of thepresent invention as described above has specific features of highspecific activity and optimum temperature and stable temperature rangesin a relatively higher temperature region, and therefore can be clearlydistinguished from the known nucleoside phosphorylases. Also, by usingsuch an enzyme for the production of nucleosides, the effects (1) and(2) as described above can be obtained. In addition, by using both ofthe enzymes of purine nucleoside phosphorylase and pyrimidinephosphorylase of the present invention, the above effect (3) can beexhibited.

UTILIZABILITY IN INDUSTRY

The process for producing nucleosides of the present invention uses anenzyme preparation derived from the cells of a microorganism belongingto thermophiles of the genus Bacillus containing a large amount ofheat-resistant nucleoside phosphorylase having high specific activity,and having high nucleoside phosphorylase activity per unit cell weight,as an enzyme source for the production of nucleosides, and is anextremely practical method which can produce objective nucleosides in agood yield with a small amount of the enzyme.

We claim:
 1. A biologically pure culture of Bacillus stearothermophilusTH 6-2 (FERM BP-2758).